A peptide is a natural or synthetic substance characterized by a specific sequence of component amino acids which are chemically linked together by amide bonds. A widely used approach to the chemical synthesis of peptides is known as the solid-phase method. (See Barany et al., Int. J. Peptide Protein Res. 30:705-39 [1987].) The feasibility of solid-phase synthesis was first demonstrated using an amino acid protected with carbobenzyloxy (Cbz) which was immobilized on a polystyrene compound via a benzyl ester linkage. (Merrifield, R. B., J. Am. Chem. Soc. 85:2149-54 [1963].) Since then, Merrifield has also used t-butyloxy-carbonyl (Boc) for N.varies. protection.
A critical step in the synthesis of peptides by the solid-phase method is the covalent attachment, or "anchoring", of the first (C-terminal) amino acid to the solid support in a specific and controlled manner. When the urethane protecting group-based synthesis schemes were employed for solid-phase peptide synthesis, there were specific problems with the carboxyl group activation methods for anchoring the C-terminal amino acid (Atherton, E. et al., J.C.S. Chem. Commun. 336-7, [1981]; Sieber, P. Tet. Lett. 28, 6147-50 [1987]; Albericio, F. and Barany, G. Int. J. Peptide Protein Res. 26, 92-7 [1985]). These methods specifically suffer from low functionalization and/or partial racemization. Although solutions to such problems have been proposed (Sieber, P. Tet. Lett. 28:6147-50 [1987]; Albericio, F. and Barany. G., Int. J. Peptide Protein Res. 26:92-7 [1985]; Kirstgen, R. et al., J. Chem. Soc., Chem. Commun., 1870-1 [1987]), none of these are entirely satisfactory for industrial scale production of amino acids anchored to solid supports.
Drawbacks of these solid phase methods using carboxyl group activation include the unavailability or expense of, and/or instability of the reagents required for carboxyl group activation. In addition, esterification via carboxyl group activation in some of these cases appears to require long reaction times for optimum yields. Finally, the ability to generalize these methods to all of the amino acids of common interest has not yet been demonstrated.
9-fluorenyl methyloxy carbonyl (Fmoc) protected amino acids have been used to prepare solid-phase bound amino acids. Racemization of the amino acid from one to about twenty percent has been observed by the currently used method for attachment of N.varies.-Fmoc amino acids to solid supports (Atherton E. et al., J.C.S. Chem. Commun. 336-337 [1981]). In another approach, amino acids are esterified directly to alkyl halides or polymeric alkyl halides without racemization. These methods require the laborious and expensive preparation of protected amino acid cesium salts. For Fmoc-amino acid cesium salts, the method (Columbo) has not been generally demonstrated, presumably due to poor solubility of some of these derivatives (e.g., Mitchell, A. R. et al., J. Org. Chem. 43:2845-52 [1978]; Wang, S. S. et al., J. Org. Chem. 42:1286-90 [1977]; Columbo, R., et al., Int. J. Peptide Protein Res. 21:118-26 [1983]). Alternate methods for production of solid support derivatives which utilize benzyl ester formation via reaction of activated carboxyl groups with benzyl alcohol derivatives in the presence of acylation catalyst have been shown to produce measurable amounts of racemization of the protected amino acid (Dhaon, M. K. et al., J. Org. Chem. 47:1962-1965 [1981]; Atherton, E. et al., J.C.S. Chem. Commun. 336-337 [1981]).
There exists a need for a general and inexpensive means for controlled and efficient anchoring of C-terminal amino acids to solid supports without significant racemization of the amino acids.